The goal of plant breeding is to combine in a single variety/hybrid various desirable traits of the parental lines. For field crops, these traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and fruit size, is important.
Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinating if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.
In Brassica, the plant is normally self sterile and can only be cross-pollinated. In self-pollinating species, such as soybeans and cotton, the male and female plants are anatomically juxtaposed. During natural pollination, the male reproductive organs of a given flower pollinate the female reproductive organs of the same flower.
Maize plants (Zea mays L.) present a unique situation in that they can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. It can self or cross pollinate. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears.
A reliable method of controlling fertility in plants would offer the opportunity for improved plant breeding. This is especially true for development of maize hybrids, which relies upon some sort of male sterility system and where a female sterility system would reduce production costs.
The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection are two of the breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. A hybrid maize variety is the cross of two such inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential. The hybrid progeny of the first generation is designated F.sub.1. In the development of hybrids only the F.sub.1 hybrid plants are sought. The F.sub.1 hybrid is more vigorous than its inbred parents. This hybrid vigor, or heterosis, can be manifested in many ways, including increased vegetative growth and increased yield.
Hybrid maize seed can be produced by a male sterility system incorporating manual detasseling. To produce hybrid seed, the male tassel is removed from the growing female inbred parent, which has been planted in alternating rows with the male inbred parent. Consequently, providing that there is sufficient isolation from sources of foreign maize pollen, the ears of the female inbred will be fertilized only with pollen from the male inbred. The resulting seed is therefore hybrid and will form hybrid plants.
The natural variation in plant development can result in plants tasseling after manual detasseling is completed. Or, a detasseler will not completely remove the tassel of the plant. In any event, the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being harvested along with the hybrid seed which is normally produced.
Alternatively, the female inbred can be mechanically detasseled by machine. Mechanical detasseling is approximately as reliable as hand detasseling, but is faster and less costly. However, most detasseling machines produce more damage to the plants than hand detasseling. Thus, no form of detasseling is presently entirely satisfactory, and a need continues to exist for alternatives which further reduce production costs and the eliminate self-pollination in the production of hybrid seed.
A reliable system of genetic male sterility would provide advantages. The laborious detasseling process can be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as a result of factors resulting from the cytoplasmic, as opposed to the nuclear, genome. Thus, this characteristic is inherited exclusively through the female parent in maize plants, since only the female provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another inbred that is not male-sterile. Pollen from the second inbred may or may not contribute genes that make the hybrid plants male-fertile. Usually seed from detasseled normal maize and CMS produced seed of the same hybrid must be blended to insure that adequate pollen loads are available for fertilization when the hybrid plants are grown and to insure diversity.
There can be other drawbacks to CMS. One is an historically observed association of a specific variant of CMS with susceptibility to certain crop diseases. This problem has discouraged widespread use of that CMS variant in producing hybrid maize.
Further, it can be appreciated that control of female fertility has advantages. Currently, once the female inbred is rendered male sterile, and the cross pollination has occurred, the male inbred plant is then physically removed since any inbred seed on the plant cannot be sold and should not be released. This adds to hybrid production the expense through the removal process. However, if the male inbred could be rendered female infertile, it would not be necessary to remove the rows of males, and any chance of inbred seed becoming available is reduced. Approximately 20 percent of acreage in producing a hybrid must be devoted to growing the male inbred. Therefore, hybrid seed is being produced on only 80% of the land being utilized for hybrid production. With female sterility in the male inbred, the male and female inbred can be grown together, with considerable cost savings in terms of land-use efficiency and in terms of field activities.
One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar, et al. However, this form of genetic male sterility requires maintenance of multiple mutant genes at separate locations within the genome and requires a complex marker system to track the genes and make use of the system convenient. Patterson also described a genic system of chromosomal translocations which can be effective, but which are complicated. U.S. Pat. Nos. 3,861,709 and 3,710,511.
Many other attempts have been made to improve on these drawbacks. For example, Fabijanski, et al., developed several methods of causing male sterility in plants (see EPO 89/3010153.8 publication No. 329,308 and PCT application PCT/CA90/00037 published as WO 90/08828). One method includes delivering into the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter. Another involves an antisense system in which a gene critical to fertility is identified and an antisense to the gene inserted in the plant. Mariani, et al. also shows several cytotoxin encoding gene sequences, along with male tissue specific promoters and mentions an antisense system. See EP 89/401,194. Still other systems use "repressor" genes which inhibit the expression of another gene critical to male sterility. PCT/GB90/00102, published as WO 90/08829.
There has been little previous incentive to control female fertility, thus little work in this area. One example is the work by De Greef et al described at European patent publication 0 412 006; U.S. Pat. No. 5,633,441. There, it was noted that female sterility was useful for producing fruit without seeds, enhanced vegetative biomass production and more flower setting within one season (p. 3/I 54-56). The system set forth there is much like the male sterility system previously employed: the plant is transformed with a female tissue specific promoter linked to a nucleotide sequence which, when expressed, disturbs development of the cell of the flower, seed, or embryo. A selectable marker is also included for selection of transformed cells (p. 4/I 1-34).
A tissue specific system for female fertility is described at DeGreef, supra, in which female sterility is induced, unlike the present invention. Again, this invention provides advantages in that the present invention provides for complete sterility, since the plant is constitutively sterile. The DeGreef system requires spraying to induce sterility, which will not be as efficient.
The disadvantage of such prior systems is that the plant is normally fertile and sterility is initiated by a variety of approaches such as using a mutant gene, tissue specific cell killing, spraying a chemical that induces sterility or the like, and are complex, difficult to use, require some detasseling, are not reliable in causing all the desired plants to be sterile, thereby allowing some inbred seed to be produced. These systems use considerable amounts of chemicals or DNA sequences which may be undesirable in a grain producing plant. They all cause sterility and fertility is restored by reversion to the native constitutively fertile state.
Here, the inventors have taken an entirely different approach. The invention allows the plant to be constitutively sterile, with fertility (not sterility) induced. This has several advantages.
Inducement of sterility can be inefficient. In male sterility, there are in excess of six to fourteen million pollen grains in one tassel that mature at different times. The inducement of sterility thus must be extremely foolproof to avoid unintentional self pollination. On the other hand, inducement of fertility need only be minimally effective since more than adequate pollen will be produced through partial restoration to achieve fertilization and increase in parent seed. Chemical treatment failure results in under production of pollen, and since pollen is normally overproduced by a wide margin, the process of this invention for production of parent seed will tolerate a treatment failure rate as high as 70% to 80% with minimal effects on yield of parent seed.
The present invention has similar advantages in producing a constitutively female sterile plant as in producing a constitutively male sterile plant. One need not be concerned with escapes that could occur when sterility is chemically induced, leaving inbred seed in a bag of hybrid seed or available for theft. A tissue specific system for female fertility is described at DeGreef, supra, in which female sterility is induced, unlike the present invention. Again, this invention provides advantages in that the present invention provides for complete sterility, since the plant is constitutively sterile. The DeGreef system requires spraying to induce sterility, which will not be as reliable.
There is no detasseling required for constitutively male sterile plants, whereas with CMS, there is only a reduction of detasseling because of the blending requirement previously described.
By having the critical gene normally "off", chemical treatment is not necessary in the large-scale production of hybrid seed, so that chemical usage (and associated expense) is minimized and the risk of treatment failure is present only in the carefully controlled, limited scale production of parent seed, where self-pollination is desired.
Maize hybrid production has been historically, and out of necessity, performed by planting a series of female rows (4-6) and male rows (1-2). This means that 20-25% of the acres in a hybrid production field are used by plants (the male parent) that are not harvested for hybrid seed, thus increasing the cost of hybrid seed on a per acre basis. In addition, current germ plasm protection requirements necessitate the destruction of the male parent after pollination. Seed that forms on the male plant represents the genotype of that inbred and is, therefore, subject to theft. With female sterility, the current invention will eliminate both the separate row requirement and the requirement to destroy the male plants. Engineered plants will not produce seeds unless the plant has been induced to do so. If that is the case, then the male parent could be interplanted with a male-sterile female parent. Alternatively, the male plant could be planted in-between certain of the female rows. These plants could be simply run-over by the harvest machinery with no fear of picking up ears (representing self-pollinations) from the male rows. Additionally, these males would not have to be destroyed as there would be no ears on the plants.
Thus, one objective of the invention is a unique variation to the method of controlling sterility by using a DNA molecule construct that enables a plant to remain sterile after transformation, with fertility, not sterility, induced via the DNA construct.
A still further object is to provide a method of mediating fertility in plants by regulating expression of the DNA molecule naturally occurring in the plant.
Yet another object is to provide a method of mediating fertility in plants by delivering the DNA molecule into a plant such that expression of the DNA molecule may be controlled.
Another object is to provide plants wherein fertility of the plants is mediated by the DNA molecule.
A further object is to use plants having fertility mediated by the DNA molecules in a plant breeding system.
Further objects of the invention will become apparent in the description and claims that follow.